Angiogenesis is a highly complex process of developing new blood vessels that involves the proliferation and migration of, and tissue infiltration by capillary endothelial cells from pre-existing blood vessels, cell assembly into tubular structures, joining of newly forming tubular assemblies to closed-circuit vascular systems, and maturation of newly formed capillary vessels. The molecular bases of many of these aspects are still not understood.
Angiogenesis is important in normal physiological processes including embryonic development, follicular growth, and wound healing, as well as in pathological conditions such as tumor growth and in non-neoplastic diseases involving abnormal neovascularization, including neovascular glaucoma (Folkman, J. and Klagsbrun, M. Science 235:442-447 (1987). Other disease states include but are not limited to, neoplastic diseases, including but not limited to solid tumors, autoimmune diseases and collagen vascular diseases such as, for example, rheumatoid arthritis, and ophthalmalogical conditions such as diabetic retinopathy, retrolental fibroplasia and neovascular glaucoma. Conditions or diseases to which persistent or uncontrolled angiogenesis contribute have been termed angiogenic dependent or angiogenic associated diseases.
One means of controlling such diseases and pathological conditions comprises restricting the blood supply to those cells involved in mediating or causing the disease or condition. For example, in the case of neoplastic disease, solid tumors develop to a size of about a few millimeters, and further growth is not possible, absent angiogenesis within the tumor. In the past, strategies to limit the blood supply to tumors have included occluding blood vessels supplying portions of organs in which tumors are present. Such approaches require the site of the tumor to be identified and are generally limited to treatment to a single site, or small number of sites. An additional disadvantage of direct mechanical restriction of a blood supply is that collateral blood vessels develop, often quite rapidly, restoring the blood supply to the tumor.
Other approaches have focused on the modulation of factors that are involved in the regulation of angiogenesis. While usually quiescent, vascular endothelial proliferation is highly regulated, even during angiogenesis. Examples of factors that have been implicated as possible regulators of angiogenesis in vivo include, but are not limited to, transforming growth factor beta (TGFβ), acidic and basic fibroblast growth factor (aFGF and bFGF), platelet derived growth factor (PDGF), and vascular endothelial growth factor (VEGF) (Klagsbrun, M. and D'Amore, P. (1991) Annual Rev. Physiol. 53: 217-239).
One growth factor of particular interest is VEGF. An endothelial-cell specific mitogen, VEGF acts as an angiogenesis inducer by specifically promoting the proliferation of endothelial cells. It is a homodimeric glycoprotein consisting of two 23 kD subunits with structural similarity to PDGF. Four different monomeric isoforms of VEGF resulting from alternative splicing of mRNA have been identified. These include two membrane bound forms (VEGF206 and VEGF189) and two soluble forms (VEGF165, and VEGF121). VEGF165 is the most abundant isoform in all human tissues except placenta.
VEGF is expressed in embryonic tissues (Breier et al., Development (Camb.) 114:521 (1992)), macrophages, and proliferating epidermal keratinocytes during wound healing (Brown et al., J. Exp. Med., 176:1375 (1992)), and may be responsible for tissue edema associated with inflammation (Ferrara et al., Endocr. Rev. 13:18 (1992)). In situ hybridization studies have demonstrated high levels of VEGF expression in a number of human tumor lines including glioblastoma multiforme, hemangioblastoma, other central nervous system neoplasms and AIDS-associated Kaposi's sarcoma (Plate, K. et al. (1992) Nature 359: 845-848; Plate, K. et al. (1993) Cancer Res. 53: 5822-5827; Berkman, R. et al. (1993) J. Clin. Invest. 91: 153-159; Nakamura, S. et al. (1992) AIDS Weekly, 13 (1)). High levels of VEGF also have been reported in hypoxia induced angiogenesis (Shweiki, D. et al. (1992) Nature 359: 843-845).
VEGF mediates its biological effect through high affinity VEGF receptors which are selectively expressed on endothelial cells during, for example, embryogenesis (Millauer, B., et al. (1993) Cell 72: 835-846) and tumor formation. VEGF receptors typically are class III receptor-type tyrosine kinases characterized by having several, typically 5 or 7, immunoglobulin-like loops in their amino-terminal extracellular receptor ligand-binding domains (Kaipainen et al., J. Exp. Med. 178:2077-2088 (1993)). The other two regions include a transmembrane region and a carboxy-terminal intracellular catalytic domain interrupted by an insertion of hydrophilic interkinase sequences of variable lengths, called the kinase insert domain (Terman et al., Oncogene 6:1677-1683 (1991)). VEGF receptors include flt-1, sequenced by Shibuya M. et al., Oncogene 5, 519-524 (1990); flk-1, sequenced by Matthews W. et al. Proc. Natl. Acad. Sci. USA, 88:9026-9030 (1991) and KDR, the human homologue of flk-1, described in PCT/US92/01300, filed Feb. 20, 1992, and in Terman et al., Oncogene 6:1677-1683 (1991).
High levels of flk-1 are expressed by endothelial cells that infiltrate gliomas (Plate, K. et al., (1992) Nature 359: 845-848), and are specifically upregulated by VEGF produced by human glioblastomas (Plate, K. et al. (1993) Cancer Res. 53: 5822-5827). The finding of high levels of flk-1 expression in glioblastoma associated endothelial cells (GAEC) suggests that receptor activity is induced during tumor formation, since flk-1 transcripts are barely detectable in normal brain endothelial cells. This upregulation is confined to the vascular endothelial cells in close proximity to the tumor. Blocking VEGF activity with neutralizing anti-VEGF monoclonal antibodies (mAbs) results in inhibition of the growth of human tumor xenografts in nude mice (Kim, K. et al. (1993) Nature 362: 841-844), suggesting a direct role for VEGF in tumor-related angiogenesis.
Various chemotherapeutic drugs also have been shown to block functions of activated, dividing endothelial cells critical to angiogenesis, or to kill such cells. Such collateral damaging effects on a genetically stable normal host cell, in addition to the chemotherapeutic agent's effect upon the tumor cells, contribute significantly to the in vivo anti-tumor efficacy of chemotherapy. However, the standard use of chemotherapeutic agents has obvious undesirable side-effects upon the normal cells of patients, limiting its use. Administration of chemotherapeutic agents in their usual doses and at usual dosage frequencies are commonly associated with side-effects, including, but not limited to, myelosuppression, neurotoxicity, cardiotoxicity, alopecia, nausea and vomiting, nephrotoxicity, and gastrointestinal toxicity. Further, patients' tumors often also develop resistance to the chemotherapeutic agents after initial exposure to the drugs.
A desirable method and composition for controlling angiogenesis should be well tolerated, have few or no side-effects, and prevent new vessel formation at sites of disease without interfering with required physiologic angiogenesis in normal sites. It should be effective and, in the case of neoplastic disease, overcome the problem of the development of drug resistance by tumor cells. In so doing, it should permit targeted therapy without the accurate identification of all disease sites. The present invention addresses many of the problems with existing materials and methods.